Tobacco rattle virus genome alterations in the Hosta hybrid 'Green Fountain' and other plants: reassortments, recombinations and deletions.

Tobacco rattle virus from a Hosta hybrid contained one RNA1 (Ho-1) and two RNA2 species (Ho-2a, Ho-2b). Whereas Ho-1 resembles TRV Al RNA1 from Alstroemerias, Ho-2a and Ho-2b resemble TRV TpO1 RNA2 from a potato field. Ho-2a has a complete RNA2-specific sequence, whereas that of Ho2-b carries a large deletion. The short RNA1-related 3' end of Ho-2a is distinct from that of Ho-1, whereas the longer one of Ho-2b is identical to that of Ho-1. TRV RNA2 molecules may apparently become associated with different TRV RNA1 molecules, from which they can acquire 3'ends of various lengths while often losing large portions of their RNA2-specific sequences.

Deletions and recombinations with the RNA1 3' ends of different tobraviruses have created a multitude of tobacco rattle virus TCM-related RNA2 species in Alstroemeria and tulip.

In vegetatively propagated Alstroemeria plants that showed pronounced stunting and necrotic leaf spots, a tobravirus infection was diagnosed in which one tobacco rattle virus (TRV, strain AL) RNA1 species was associated with seven different RNA2 species. The latter differed considerably in size and in the types of their 3' RNA1-related sequences. The 5' RNA2-specific part of all these RNA2 molecules showed almost 100% sequence identity with that of RNA2 of the TRV isolate TCM from tulip, but in some of these RNA2 molecules it was shorter than in the TCM isolate, whereas in others it was longer. One of the TRV AL RNA2 molecules, i.e. TC3'PE-a, contained the full set of three full-length RNA2-specific ORFs (ORF2a, -2b and -2c), whereas the previously analysed TCM sequence contained only ORF2a and -2b. In four of these TRV AL RNA2 molecules, i.e. those that had a relatively short RNA2-specific part, the 3' end was identical to that of the cognate TRV AL RNA1, but in the other three, which had a long RNA2-specific part, it was closely related to that of pea early browning virus (PEBV) RNA1, which was not detected in the infected plants. A comparison with previously described TRV/PEBV RNA2 recombinants suggested that the various TRV AL RNA2 molecules may represent various steps and side steps in an evolutionary process, which is apt to open the wide host range of TRV also to PEBV-derived RNA2 species.

NMR structure of a classical pseudoknot: interplay of single- and double-stranded RNA.

Pseudoknot formation folds the 3' ends of many plant viral genomic RNAs into structures that resemble transfer RNA in global folding and in their reactivity to transfer RNA-specific proteins. The solution structure of the pseudoknotted T arm and acceptor arm of the transfer RNA-like structure of turnip yellow mosaic virus (TYMV) was determined by nuclear magnetic resonance (NMR) spectroscopy. The molecule is stabilized by the hairpin formed by the 5' end of the RNA, and by the intricate interactions related to the loops of the pseudoknot. Loop 1 spans the major groove of the helix with only two of its four nucleotides. Loop 2, which crosses the minor groove, interacts closely with its opposing helix, in particular through hydrogen bonds with a highly conserved adenine. The structure resulting from this interaction between the minor groove and single-stranded RNA at helical junctions displays internal mobility, which may be a general feature of RNA pseudoknots that regulates their interaction with proteins or other RNA molecules.

Pseudoknots: a new motif in the RNA game.

In the last few years a novel RNA folding principle called pseudoknotting has emerged. Originally discovered in noncoding regions of plant viral RNAs, pseudoknots now appear to be a widespread structural motif in a number of functionally different RNAs. These structural elements are part of tRNA-like structures and are involved in folding catalytic sites of ribozymes. They increase the efficiency of ribosomal frameshifting or can serve as specific binding sites for regulatory proteins.

Structural parameters affecting the kinetics of RNA hairpin formation.

There is little experimental knowledge on the sequence dependent rate of hairpin formation in RNA. We have therefore designed RNA sequences that can fold into either of two mutually exclusive hairpins and have determined the ratio of folding of the two conformations, using structure probing. This folding ratio reflects their respective folding rates. Changing one of the two loop sequences from a purine- to a pyrimidine-rich loop did increase its folding rate, which corresponds well with similar observations in DNA hairpins. However, neither changing one of the loops from a regular non-GNRA tetra-loop into a stable GNRA tetra-loop, nor increasing the loop size from 4 to 6 nt did affect the folding rate. The folding kinetics of these RNAs have also been simulated with the program 'Kinfold'. These simulations were in agreement with the experimental results if the additional stabilization energies for stable tetra-loops were not taken into account. Despite the high stability of the stable tetra-loops, they apparently do not affect folding kinetics of these RNA hairpins. These results show that it is possible to experimentally determine relative folding rates of hairpins and to use these data to improve the computer-assisted simulation of the folding kinetics of stem-loop structures.

PseudoBase: structural information on RNA pseudoknots.

PseudoBase is a database containing structural, functional and sequence data related to RNA pseudo-knots. It can be reached at http://wwwbio.LeidenUniv.nl/ approximately Batenburg/PKB.html. For each pseudoknot, thirteen items are stored, for example the relevant sequence, the stem positions of the pseudoknot, the EMBL accession number of the sequence and the support that can be given regarding the reliability of the pseudo-knot. Since the last publication, information on sizes of the stems and the loops in the pseudoknots has been added. Also added are alternative entries that produce surveys of where the pseudoknots are, sorted according to stem size or loop size.

A pH-jump approach for investigating secondary structure refolding kinetics in RNA.

It has been shown that premature translation of the plasmid-mediated toxin in hok/sok of plasmid R1 and pnd/pndB of plasmid R483 is prevented during transcription of the hok and pnd mRNAs by the formation of metastable hairpins at the 5'-end of the mRNA. Here, an experimental approach is presented, which allows the accurate measurement of the refolding kinetics of the 5'-end RNA fragments in vitro without chemically modifying the RNA. The method is based on acid denaturation followed by a pH-jump to neutral pH as a novel way to trap kinetically favoured RNA secondary structures, allowing the measurement of a wide range of biologically relevant refolding rates, with or without the use of standard stopped-flow equipment. The refolding rates from the metastable to the stable conformation in both the hok74 and pnd58 5'-end RNA fragments were determined by using UV absorbance changes corresponding to the structural rearrangements. The measured energy barriers showed that the refolding path does not need complete unfolding of the metastable structures before the formation of the final structures. Two alternative models of such a pathway are discussed.

Aminoacylation of 3' terminal tRNA-like fragments of turnip yellow mosaic virus RNA: the influence of 5' nonviral sequences.

The present model of the L-shaped tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA encompasses 82 nucleotides. A previous kinetic study on 3' terminal TYMV RNA fragments that contain the tRNA-like structure and a 5' nonviral GGGAGA sequence, suggested that viral sequences upstream of the tRNA-like domain, i.e., upstream of nucleotide 82, increase the rate of aminoacylation (Dreher et al. (1988) Biochimie 70, 1719-1727). Here we report an increase in the aminoacylation rate when the number of nonviral nucleotides at the 5' end of TYMV RNA transcripts was reduced. The influence of these 5' proximal nonviral sequences on the conformation of the RNA molecule was investigated by structure mapping experiments. A structure that deviates from the tRNA-like structure was found in some of the transcripts. The formation of this alternative structure is dependent upon: (1) the nature and number of the nonviral nucleotides; (2) the number and secondary structure of viral nucleotides between the nonviral nucleotides and the tRNA-like domain. Footprinting experiments with valyl-tRNA synthetase from yeast suggest that the enzyme does not recognize the alternative structure.

The computer simulation of RNA folding pathways using a genetic algorithm.

A procedure for simulating the RNA folding process using the principles of genetic algorithm is proposed. The method allows one to simulate a folding pathway of RNA, including such processes as disruption of temporarily formed structures, the folding of a molecule during its synthesis and pseudoknot formation. The simulations are able to predict functional metastable foldings and kinetically driven transitions to more stable structures. The analysis of free energies for intermediate foldings allows estimation of the ranges of kinetic refolding barriers and suggests that in some RNAs the selective evolutionary pressure suppresses the possibilities for alternative structures that could form in the course of transcription. It is shown that the folding pathway simulation can result in structure predictions that are more consistent with phylogenetically proven structures than minimum energy solutions. This suggest that RNA folding kinetics is very important for the formation of functional RNA structures. Therefore, apart form its value for predictions of RNA structures, the proposed computer simulations can be a powerful tool in the studies of RNA folding features.

Dynamic competition between alternative structures in viroid RNAs simulated by an RNA folding algorithm.

The folding pathways of viroid RNAs were studied using computer simulations by the genetic algorithm for RNA folding. The folding simulations were performed for PSTVd RNAs of both polarities, using the wild-type sequence and some previously known mutants with suggested changes in the stable or metastable structures. It is shown that metastable multihairpin foldings in the minus strand replicative intermediates are established due to the specific folding pathway that ensures the absence of the most stable rod-like structure. Simulations of the PSTVd minus strand folding during transcription reveal a metastable hairpin, formed in the left terminal domain region of the PSTVd. Despite high sequence variability, this hairpin is conserved in all known large viroids of both subgroups of PSTVd type, and is presumably necessary to guide the folding of the HPII hairpin which is functional in the minus strand. The folding simulations are able to demonstrate the changes in the balance between metastable and stable structures in mutant PSTVd RNAs. The stable rod-like structure of the circular viroid (+) RNA is also folded via a dynamic folding pathway. Furthermore, the simulations show that intermediate steps in the forced evolution of a shortened PSTVd replicon may be reconstructed by a mechanistic model of different folding pathway requirements in plus- and minus-strand RNAs. Thus the formation of viroid RNA structure strongly depends on dynamics of competition between alternative RNA structures. This also suggests that the replication efficiency of viroid sequences may be estimated by a simulation of the folding process.

Mutational analysis of the pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA. Aminoacylation efficiency and RNA pseudoknot stability.

Site-directed mutations were introduced in the connecting loops and one of the two stem regions of the RNA pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA. The kinetic parameters of valylation for each mutated RNA were determined in a cell-free extract from wheat germ. Structure mapping was performed on most mutants with enzymic probes, like RNase T1, nuclease S1 and cobra venom ribonuclease. An insertion of four A residues in the four-membered connecting loop L1 that crosses the deep groove of the pseudoknot reduces aminoacylation efficiency. Deletions up to three nucleotides do not affect aminoacylation or RNA pseudoknot formation. Deletion of the entire loop abolishes aminoacylation. Although elimination of the pseudoknot is presumed, this could not be demonstrated. Unlike the mutations in loop L1, all mutations in the three-membered connecting loop L2 that crosses the shallow groove of the RNA pseudoknot decrease the aminoacylation efficiency considerably. Nonetheless, the RNA pseudoknot is still present in most mutated RNAs. These results indicate that a number of mutations can be introduced in both loops without abolishing aminoacylation. Results obtained with the introduction of mismatches and A.U base-pairs in stem S1 of the pseudoknot, containing three G.C base-pairs in wild-type RNA, indicate that the pseudoknot is only marginally stable. Our estimation of the gain of free energy due to the pseudoknot formation is at most 2.0 kcal/mol. The pseudoknot structure can, however, be stabilized upon binding the valyl-tRNA synthetase.

Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting.

RNA pseudoknots play important roles in many biological processes. In the simian retrovirus type-1 (SRV-1) a pseudoknot together with a heptanucleotide slippery sequence are responsible for programmed ribosomal frameshifting, a translational recoding mechanism used to control expression of the Gag-Pol polyprotein from overlapping gag and pol open reading frames. Here we present the three-dimensional structure of the SRV-1 pseudoknot determined by NMR. The structure has a classical H-type fold and forms a triple helix by interactions between loop 2 and the minor groove of stem 1 involving base-base and base-sugar interactions and a ribose zipper motif, not identified in pseudoknots so far. Further stabilization is provided by a stack of five adenine bases and a uracil in loop 2, enforcing a cytidine to bulge. The two stems of the pseudoknot stack upon each other, demonstrating that a pseudoknot without an intercalated base at the junction can induce efficient frameshifting. Results of mutagenesis data are explained in context with the present three-dimensional structure. The two base-pairs at the junction of stem 1 and 2 have a helical twist of approximately 49 degrees, allowing proper alignment and close approach of the three different strands at the junction. In addition to the overwound junction the structure is somewhat kinked between stem 1 and 2, assisting the single adenosine in spanning the major groove of stem 2. Geometrical models are presented that reveal the importance of the magnitude of the helical twist at the junction in determining the overall architecture of classical pseudoknots, in particular related to the opening of the minor groove of stem 1 and the orientation of stem 2, which determines the number of loop 1 nucleotides that span its major groove.

Acetylation of ribosomal protein S5 affected by defects in the central pseudoknot in 16S ribosomal RNA?

We have analyzed the ribosomal protein profile of Escherichia coli 30S subunits with the mutation C18A in the central pseudoknot of their 16S ribosomal RNA. This mutation was shown to inhibit translational activity in vivo and to affect ribosome stability in vitro. The majority of the mutant 30S particles were present as free subunits in which a reproducible decrease in amount of proteins S1, S2, S18 and S21 was observed. The protein gels also showed the appearance of a satellite band next to S5. This band reacted with anti-S5 antibodies and had a slightly increased positive charge. The simplest interpretation of these findings, also considering published data, is that the satellite band is S5 with a non-acetylated N-terminal alanine. Underacetylation of S5 due to mutations in the 16S rRNA implies that the modification is performed on the ribosome.

Methylation of the conserved A1518-A1519 in Escherichia coli 16S ribosomal RNA by the ksgA methyltransferase is influenced by methylations around the similarly conserved U1512.G1523 base pair in the 3' terminal hairpin.

An in vitro system developed for the site-specific mutagenesis of 16S rRNA of Escherichia coli ribosomes was used to make five mutations around the highly conserved U1512.G1523 base pair in the 3' terminal hairpin. Each of the mutant RNAs was reconstituted with a complete mixture of 30S proteins to yield 30S ribosomal particles, which were tested for the ability of the ksgA methylase to form m6(2)A1518 and m6(2)A1519. Dimethylation of A1518 and A1519 in the hairpin loop was inhibited 20-80% by the mutations. The results indicate that G1523 and C1524 in the stem are important determinants for the dimethylation of A1518 and A1519 in the loop. Either the enzyme recognition region extends that far or the effect of mutations in the stem are propagated in some manner to the loop. The conserved U.G base pair does not of itself appear to play a major role in ksgA methylase recognition.

Efficient transcription of the tRNA-like structure of turnip yellow mosaic virus by a template-dependent and specific viral RNA polymerase obtained by a new procedure [corrected].

The RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus (TYMV) was isolated by a simple, new method. An active, template-dependent and specific enzyme was obtained. Although the genomic RNA of TYMV could not be transcribed completely during an in vitro RdRp assay, a complete double-stranded product was obtained when a 3' terminal RNA fragment of 83 nucleotides was used as a template. The reaction product was identified as being of negative polarity by complete digestion with ribonuclease T1. Antibodies directed to part of the N-terminal (Ab140) or C-terminal (Ab66) in vitro autocleavage products of the large non-structural polyprotein of TYMV, could both partially inhibit RdRp activity. Further purification of the RdRp preparation by ion-exchange chromatography resulted in two activity peaks with different protein compositions. Both peak fractions retained high specificity for transcription of TYMV RNA. A protein of approximately 115 kDa was detected by both Ab140 and Ab66.

3-D graphics modelling of the tRNA-like 3'-end of turnip yellow mosaic virus RNA: structural and functional implications.

The tRNA-like structure of the aminoacylatable 3'-end of turnip yellow mosaic virus (TYMV) RNA was submitted to 3-D graphics modelling. A model of this structure has been inferred previously from both biochemical results and sequence comparisons which presents a new RNA folding feature, the "pseudoknot". It has been verified that this structure can be constructed without compromising accepted RNA stereochemical rules, namely base stacking and preferential 3'-endo sugar pucker. The model has aided interpretation of previous structural mapping experiments using chemical and enzymatic probes, and new accessibilities of residues could be predicted and tested. Pseudoknots have been considered as potential splice sites because they form antiparallel helical segments in a single RNA molecule. We have examined this possibility with the constructed 3-D model and could verify the hypothesis on a structural basis. The model presents a striking similarity with canonical tRNA and allows a valuable comparison between the protection patterns of yeast tRNA(Val) and tRNA-like viral RNA by cognate yeast valyl-tRNA synthetase against structural probes.

Molecular characterization of isolates of anagyris vein yellowing virus, plantago mottle virus and scrophularia mottle virus -- comparison of various approaches for tymovirus classification.

The complete nucleotide sequences were determined for the genomic RNAs of three tymoviruses, i.e. isolates of anagyris vein yellowing virus (AVYV), plantago mottle virus (PlMoV) and scrophularia mottle virus (SrMV) which are all serologically closely related to ononis yellow mosaic virus (ibid) and to Nemesia ring necrosis virus (NeRNV), a recently described recombinant virus which is widely spread in commercially grown ornamental plant species belonging to the Scrophulariaceae. Total nucleotide and coat protein amino acid sequence identities revealed similar groupings in the genus tymovirus as serological studies did. The latter, however, tended to suggest much closer relationships than the molecular data and may fail to recognise the distinctiveness of new tymovirus species. The usefulness of various species demarcation criteria for the classification of tymoviruses is discussed.